The present invention relates generally to biosensors, and more particularly, to a fluorescence sensor that utilizes a capillary waveguide.
Fluorescence sensors having a capillary as a light guiding device are effective analytical tools used to measure the concentrations of different analytes as well as for detecting the presence of nucleic acids such as DNA. Particularly useful applications include DNA sequencing, genetic analysis, medical diagnosis and forensics. The basic approach is to monitor a fluid sample containing the target analyte or nucleic acids by illuminating a region of a capillary core with a laser and measuring the induced fluorescence as molecules move through the illuminated region.
One type of fluorescence sensor utilizes a direct detection process, wherein the target fluid sample is provided with a fluorescent marker prior to injection of the sample into the capillary. Another type of fluorescence sensor utilizes an indirect detection process, wherein a surface of the capillary that will come in contact with the target sample is coated with a layer of probe molecules.
Nucleic acid based sensors offer a high degree of selectivity and stability. Detection is based on specific hybridization between a single-stranded nucleic acid oligonucleotide “probe” sequence and the sample “target” sequence to be detected. The probe is typically immobilized on a substrate, such as an optical fiber or a planar waveguide. In fluorescence based sensors, target sequences are tagged with fluorescent molecules. In situ hybridization, standard Watson-Crick base pairing for strand annealing, allows detection of the targeted species. Denaturing, through elevated temperatures or through the use of denaturants, allows the sensor to be used for repeated detection.
A conventional fluorescence sensor 100, as depicted in FIG. 1, employs a coaxial optical geometry for excitation and collection. Such first generation sensors used an unclad quartz rod 102 typically 0.5 to 1 mm in diameter and several centimeters in length. The outer surface of the rod 102 is coated with a probe sequence. A light energy source (not shown) is positioned perpendicularly to the quartz rod 102 and directs excitation light 104 to a dichroic beam splitter 106, which redirects the light longitudinally into a proximal end 108 of the rod, which is immersed in a fluid sample 110 containing the target sequence. The fluorescence emission 114 is collected from the proximal end 108 after passing through a band pass optical filter 116.
The fluorescence captured by the guided modes of the optical rod arrangement shown in FIG. 1 is critically dependent on the numerical aperture (NA) of the excitation and collection optics. Matching of the optical system NA to the sensor NA, which is determined by the index of the refraction of the core and the sample, is critical to successful functioning of the sensor. Traditionally, for ease of use, a backward scheme is preferred such that the fluorescence is collected from the proximal end surface of the fiber contrary to the preferred collection from the distal end surface.
Fiber optic sensors have gone through an evolution over the last two decades. Most designs have sought to seek improvement in sensor sensitivity through the use of tapered distal fiber ends, while others have introduced a tapered region in the center of a longer length of fiber. The tapered designs have faced the problem of mismatch between the V-number of the optical fiber in the sensor and guiding regions. To overcome some of these difficulties single mode fibers with etched claddings, exposing a longitudinal section have been proposed. Other sensors have utilized planar waveguides, fiber array imaging, and optical microcavities for containing optical wave fields.
In the last decade silica micro-capillaries have been fast replacing optical fibers in the development of analytical sensors for use in various applications. The capillary provides a unique combination of supporting both fluid flow and optical light propagation. In particular, micro-capillaries have found extensive use in DNA sequencing using gel electrophoresis. For biosensor applications, the probe sequence(s) can be covalently bonded to the interior surface of the capillary to provide detection of target sequence(s) flowing through the capillary. Coating on the interior surface also protects against accidental damage during handling.
In general, the sensitivity of fluorescence based sensors may be specified in two ways: first, by the lowest concentration of detectable target; and second, by tracking of small changes in the target concentration. In the first case, the limit is determined by system electronics, while in the second case, the limit is dependent on the stability of the various system components, such as the power stability of the excitation source. In practice, however, the limit of detection is determined by the amount of stray light bleeding into the emission band of the target species. Optimal design based on a combination of optical filtering, high speed optics and separation of excitation and collection paths is necessary to achieve limits approaching concentrations below ng/ml.
FIGS. 2a-2d illustrate some of the other prior art optical configurations which are used in capillary based fluorescence sensors. Traditionally, a coaxial arrangement, as shown in FIG. 2a, is utilized where the surface molecules are illuminated by evanescent wave field excitation (EWFE) and a portion of fluorescence which tunnels into the guided modes (FETGM) of the capillary wall is captured at either (or both) ends of the capillary. This configuration, while providing uniform illumination of the hybridized molecules along the entire capillary length has the highest excitation background and requires considerable optical filtering. Additionally, this arrangement does not allow independent optimization of excitation and emission geometries.
FIG. 2b illustrates the arrangement used for capillary gel electrophoresis. In this arrangement, 90-degree separation between the excitation and fluorescence emission paths (FPFE) is essential to achieving sensitivities approaching 10−12 M.
FIG. 2c shows a forward transmitting geometry which has been used for detecting CO2 changes through either fluorescence or absorption. This arrangement typically uses grating couplers, printed on the outer surface of the capillary, for exciting higher order modes in the capillary wall and for interrogating the guided fluorescence emission. Prism couplers are also typically employed for selectively coupling energy into and out from the planar waveguides.
FIG. 2d illustrates an orthogonal arrangement. Excitation energy is launched into the waveguide at right angles to the capillary surface and the integrated fluorescence energy tunneled into the guided modes of the capillary wall is collected at the capillary end face. This arrangement does not provide uniform and efficient illumination of the hybridized target molecules along the entire capillary length.
It would be desirable to provide a simple biosensor without the aforementioned drawbacks. In particular, it would be desirable to provide an accurate capillary waveguide fluorescence device that is relatively compact, simple in construction and requires a smaller volume of target sample.